Journal Article

Modulation of 14-3-3 Protein Interactions with Target Polypeptides by Physical and Metabolic Effectors

Gurdeep S. Athwal, Christian R. Lombardo, Joan L. Huber, Shane C. Masters, Haian Fu and Steven C. Huber

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 41, issue 4, pages 523-533
Published in print April 2000 | ISSN: 0032-0781
Published online April 2000 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/41.4.523
Modulation of 14-3-3 Protein Interactions with Target Polypeptides by Physical and Metabolic Effectors

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  • Molecular and Cell Biology
  • Plant Sciences and Forestry

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The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in KD. At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

Keywords: AMP binding; BIAcore; Hydrolytic activity; Metal binding; Protein:protein interaction; Spinacia oleracea

Journal Article.  0 words. 

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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